Cellular growth and differentiation appear to be initiated, promoted, maintained and regulated by a multiplicity of stimulatory, inhibitory and synergistic factors and hormones. The alteration and/or breakdown of the cellular homeostasis mechanism seems to be a fundamental cause of growth related diseases, including neoplasia. Growth modular factors are implicated in a wide variety of pathological and physiological processes including signal transduction, cell communication, growth and development, embryo genesis, immune response, hematopoiesis cell survival and differentiation, inflammation, tissue repair and remodeling, atherosclerosis and cancer.
Epidermal growth factor (EGF), transforming growth factor alpha (TGFα), betacellulin, amphiregulin, and vaccinia growth factor among other factors are growth and differentiation modulatory proteins induced by a variety of cell types either under normal physiological conditions or in response to exogenous stimuli.
These peptide growth factors influence wound cells through autocrine and paracrine mechanisms. They also play important roles in normal wound healing in tissues such as skin, cornea and gastrointestinal tract and they all share substantial amino acid sequence homology including the conserved placement of three intra-chain disulfide bonds. In addition, all the factors of this family bind to a 170,000 molecular weight transmembrane glycoprotein receptor and activate the tyrosine kinase activity in the receptors cytoplasmic domain (Buhrow, S. A. et al., J. Bio. Chem., 8:7824–7826 (1983).
The receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells and epithelial cells of the gastrointestinal tract. These peptide growth factors are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. These growth factors have also been implicated in both the stimulation of growth and differentiation of certain cells.
A protein which migrates as a train of spots with an average mass of 85 kda has been disclosed (Bhalerao, et al., J. Bio. Chem., 279(27):16385–16294 (1995)). This protein has been designated as “p85”. The full length cDNA contains an open reading frame of 1,677 base pairs encoding a protein of 559 amino acids. Computer analysis of the deduced primary amino acid sequence reveals a hydrophobic signal peptide characteristic of a secreted protein. Motif analysis did not identify features typical for known protein families. The message of 1.9 kb is expressed in various tissues such as liver, heart, lungs, etc., whereas a splice variant was present in embryonic cartilage and skin. The corresponding gene for p85 (called ECM1 for extracellular matrix protein 1), maps on chromosome 3 of the mouse in a region containing several loci involved in skin development disorders. p85 was originally identified as a novel secreted protein of the mouse stromal osteogenic cell line, MN7.
The gene maps to chromosome 3, just distal to GBA in a region containing at least 3 known mutations affecting skin: FT (flaky tail), SOC (soft coat), and MA (matted). This suggests that the p85 gene may represent a candidate for any of these mutations. In particular, mice with SOC have abnormalities in the epidermis, hair bowl, whiskers and display a clumping of the hairs of the coat (Green, M. C. (1989)) in genetic variance and strains of the laboratory mouse (Lyon, M. F. and Searle, A. G., EDS) pp. 12–403, Oxford University Press, Oxford, all of which is consistent with the known expression pattern of ECM1. Correlation of SOC and ECM1 would provide important information in the elucidation of the in vivo function of p85.
The localization of ECM1 is also interesting from another standpoint. The ECM1 region shares linkage homology to human chromosome 1q21 (O'Brien, S. J. and Graves, M. J. A. (1991), Cytogenet. Cell Genet. 58:1124–1151), a region that contained a cluster of 3 families of genes involved in epidermal differentiation (Volz, A. et al., Genomics, 18:92–99 (1993)). One family includes the proteins loricrin, involucrin and a small proline-rich protein. These proteins are closely associated in the formation of the cornified cell envelope in the uppermost layers of the epidermis (Yoneda, K. et al., J. Biol. Chem., 267:18060–18066 (1992)). Each of these genes contains a region of short tandem peptide repeats that have been partially conserved during evolution (Steinert, P. M. et al., J. Biol. Macromol., 13:130–139 (1991)). A recent report demonstrated that the mouse homologue of loricrin maps to mouse chromosome 3 in apparent close proximity to ECM1 (Rothnagel, J. A. et al., Genomics, 23:450–456 (1994)).
The second group includes several members of the S100 family of small calcium-binding proteins. These proteins contain 2 calcium binding domains with the EF-hand motif, are highly homologous at the amino acid sequence level, and have a similar gene organization.
The third family localized to human 1q21 includes profilagrin and trichohyalin (Lee, S. C. et al., J. Invest. Dermatol., 100:65–69 (1993)). These genes appear to be fused genes containing at the 5′ end two EF-hand calcium finding motifs like those of the S100 family, and tandem repeats that are characteristic of the cornified cell envelope family (Markova, N. G., Mol. Cell. Biol., 13:613–625 (1993)). The mouse profilagrin locus has recently been mapped to chromosome 3 (Rothnagel, J. A. et al., Genomics, 23:450–456 (1994)).
The close physical linkage of these genes and the striking similarity in their organizations has been suggested to be the result of a common evolution (Bakendorf, C. and Hohl, D., Nature Genet., 2:91 (1992)). It has been suggested that some of these genes may share common regulatory regions and may function in concert during the final steps of epidermal differentiation (Rosenthal, D. S. et al., J. Invest. Dermatol., 98:343–350 (1992)).